Template:IONIS Paris/Notebook/28-05-15

Amplification of pSB1A2 (part BBa_J61100) into E.coli DH5 alpha

Plate preparation

Aim: preparation of selective LB agar plate

Add 40 ml of LB agar into a Falcon tube (50ml) + 40µl antibiotic For 20 ml / plate (2 plates)

Agar plates preparation
Plasmid	Antibiotic	Number of plate	Remarks
pSB1A2
	Ampicillin	1	50 µl of transformed cells
	Ampicillin	1	20 µl of transformed cells
Control negative
	Amplicilin	1	50 µl of transformed cells

Antibiotics concentration:

Ampicilin: 50 µg.ml^-1 diluted 1/1000

Cell transformation

E.coli DH5 alpha (NEB5 alpha competent coli)
Stock: 6 tubes 0.20 ml / tube; 50 pg.µl^-1
Aliquot of 50 µl of competent E.coli / transformation
100 µl remaining competent cells have been put at -80°C again (loss of efficiency)

Into aliquots, added plasmids or nothing (control) as following:
Tube n°	Reagent added	Volume (µl)
1	pSB1A2	1
2	Control negative	--

Incubation on ice, 30 min
Heat shock exactly 42°C, 30 seconds. Do not mix
Keep on ice for 5 min, do not mix
400 µl of LB in sterile condition without antibiotic, pipette up and down gently
Incubation 37°C, 120 rpm, 1 hour
Centrifuge, 4000 rpm, 5 min
Remove and discard 300 µl of supernatant; resuspend gently using a pipette the remaining 150 µl
Plate bacteria following the table about plate preparation
Incubate at 37°C, O/N

Expected results:
No colonies into the control plate
Many colonies into the other plate

Plasmid digestion (pSB1C3)

Aim: Extraction of the biobrick’s backbone for Gibson Assembly

Into aliquots, added plasmids or nothing (control) as following:
Tube	pSBC3
Water	16 µL
Buffer 2.1	2 µL
Plasmid pSB1C3	1 µL
Enzyme EcoRI	0.5 µL
Enzyme PstI	0.5 µL

NEB double digestion Buffer 2.1:

EcoRI: 100%
PstI: 75%

37°C, 1h10
Storage: -20°C

Electrophoresis

Aim: check for digestion

1 gel for purification: 110ml TAE 1X + 1,1g agarose + 140µl BET
Sample (purification): 20µl of digestion product+ 2µl of loading dye (6x)
Sample (quantification): 1µl of DNA + 2 µl of loading dye (6x) + 9µl of water MQ
Ladder: 1 µl of 2log ladder (1µg/µl) + 2 µl loading dye (6x) + 9 µl water MQ
110 V, 45 min

Expected results

Results

Liquid culture

Antibiotics concentration:
Tube 1 & 2: 3 mL LB + 3 µL Cam; colonies from “1µL” plate of Terminator T7 transformation
Tube 3 & 4: 3 mL LB + 3 µL Amp; colonies from “50µL” plate of bFos
Tube 5 & 6: 3 mL LB + 3 µL Amp; colonies from “50µL” plate of bJun
Tube 7 & 8: 3 mL LB
37°C, 120 rpm, O/N

28 May 15